Search

Heat shock protein 27 (Hsp27) is emerging as a promising therapeutic target for treatment of various cancers. Although the role of Hsp27 in protection from stress-induced intrinsic cell death has been relatively well studied, its role in Fas (death domain containing member of the tumor necrosis factor receptor superfamily)-induced apoptosis and cell proliferation remains underappreciated. Here, we show that Hsp27 silencing induces dual coordinated effects, resulting in inhibition of cell proliferation and sensitization of cells to Fas-induced apoptosis through regulation of PEA-15 (15-kDa phospho-enriched protein in astrocytes). We demonstrate that Hsp27 silencing suppresses proliferation by causing PEA-15 to bind and sequester extracellular signal-regulated kinase (ERK), resulting in reduced translocation of ERK to the nucleus. Concurrently, Hsp27 silencing promotes Fas-induced apoptosis by inducing PEA-15 to release Fas-associating protein with a novel death domain (FADD), thus allowing FADD to participate in death receptor signaling. Conversely, Hsp27 overexpression promotes cell proliferation and suppresses Fas-induced apoptosis. Furthermore, we show that Hsp27 regulation of PEA-15 activity occurs in an Akt-dependent manner. Significantly, Hsp27 silencing in a panel of phosphatase and tensin homolog on chromosome 10 (PTEN) wild-type or null cell lines, and in LNCaP cells that inducibly express PTEN, resulted in selective growth inhibition of PTEN-deficient cancer cells. These data identify a dual coordinated role of Hsp27 in cell proliferation and Fas-induced apoptosis via Akt and PEA-15, and indicate that improved clinical responses to Hsp27-targeted therapy may be achieved by stratifying patient populations based on tumor PTEN expression. [ABSTRACT FROM AUTHOR]

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid-liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Treatment-induced adaptive pathways converge to support androgen receptor (AR) reactivation and emergence of castration-resistant prostate cancer (PCa) after AR pathway inhibition (ARPI). We set out to explore poorly defined acute adaptive responses that orchestrate shifts in energy metabolism after ARPI and identified rapid changes in succinate dehydrogenase (SDH), a TCA cycle enzyme with well-known tumor suppressor activity. We show that AR directly regulates transcription of its catalytic subunits (SDHA, SDHB) via androgen response elements (AREs). ARPI acutely suppresses SDH activity, leading to accumulation of the oncometabolite, succinate. Succinate triggers calcium ions release from intracellular stores, which in turn phospho-activates the AR-cochaperone, Hsp27 via p-CaMKK2/p-AMPK/p-p38 axis to enhance AR protein stabilization and activity. Activation of this pathway was seen in tissue microarray analysis on prostatectomy tissues and patient-derived xenografts. This adaptive response is blocked by co-targeting AR with Hsp27 under both in vitro and in vivo studies, sensitizing PCa cells to ARPI treatments., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Paternally expressed gene 10 ( PEG10 ) has been associated with neuroendocrine muscle-invasive bladder cancer (MIBC), a subtype of the disease with the poorest survival. In this work, we further characterized the expression pattern of PEG10 in The Cancer Genome Atlas database of 412 patients with MIBC, and found that, compared with other subtypes, PEG10 mRNA level was enhanced in neuroendocrine-like MIBC and highly correlated with other neuroendocrine markers. PEG10 protein level also associated with neuroendocrine markers in a tissue microarray of 82 cases. In bladder cancer cell lines, PEG10 expression was induced in drug-resistant compared with parental cells, and knocking down of PEG10 resensitized cells to chemotherapy. Loss of PEG10 increased protein levels of cell-cycle regulators p21 and p27 and delayed G 1 -S-phase transition, while overexpression of PEG10 enhanced cancer cell proliferation. PEG10 silencing also lowered levels of SLUG and SNAIL, leading to reduced invasion and migration. In an orthotopic bladder cancer model, systemic treatment with PEG10 antisense oligonucleotide delayed progression of T24 xenografts. In summary, elevated expression of PEG10 in MIBC may contribute to the disease progression by promoting survival, proliferation, and metastasis. Targeting PEG10 is a novel potential therapeutic approach for a subset of bladder cancers., (©2020 American Association for Cancer Research.)

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.

Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Purpose: Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signaling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model., Experimental Design: Gene silencing of a luciferase reporter gene in the PC-3M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models., Results: LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. LNP CLU-siRNA potently suppressed CLU and AR ASO-induced CLU and AKT and ERK phosphorylation in ENZ-R LNCaP cells in vitro, more potently inhibiting ENZ-R cell growth rates and increased apoptosis when compared with AR-ASO monotherapy. In subcutaneous ENZ-R LNCaP xenografts, combinatory treatment of LNP CLU-siRNA plus AR-ASO significantly suppressed tumor growth and serum PSA levels compared with LNP LUC-siRNA (control) and AR-ASO., Conclusions: LNP siRNA can silence target genes in vivo and enable inhibition of traditionally non-druggable genes like CLU and other promising cotargeting approaches in ENZ-R CRPC therapeutics., (©2015 American Association for Cancer Research.)

Non-small cell lung cancer (NSCLC) is the most frequent cause of death from cancer worldwide. Despite the availability of active chemotherapy regimens and EGFR tyrosine kinase inhibitors, all advanced patients develop recurrent disease after first-line therapy. Although Hsp27 is a stress-induced chaperone that promotes acquired resistance in several cancers, its relationship to treatment resistance in NSCLC has not been defined. Understanding adaptive responses of acquired resistance will help guide new strategies to control NSCLC. Hsp27 levels were evaluated in an HCC827 erlotinib-resistant-derived cell line (HCC-827Resistant), and sensitivity to erlotinib was examined in Hsp27-overexpressing A549 cells. The role of Hsp27 in both erlotinib and cytotoxic treatment resistance was evaluated in HCC-827 and A549 NSCLC cells using the Hsp27 antisense drug OGX-427. The effect of OGX-427 in combination with erlotinib was also assessed in mice bearing A549 xenografts. Hsp27 is induced by erlotinib and protects NSCLC cells from treatment-induced apoptosis, whereas OGX-427 sensitizes NSCLC cells to erlotinib. Interestingly, increased resistance to erlotinib was observed when Hsp27 was increased either in HCC827 erlotinib-resistant or overexpressing A549 cells. Combining OGX-427 with erlotinib significantly enhanced antitumor effects in vitro and delayed A549 xenograft growth in vivo. OGX-427 also significantly enhanced the activity of cytotoxic drugs used for NSCLC. These data indicate that treatment-induced Hsp27 contributes to the development of resistance, and provides preclinical proof-of-principle that inhibition of stress adaptive pathways mediated by Hsp27 enhances the activity of erlotinib and chemotherapeutics., (©2015 American Association for Cancer Research.)

Incurable castration-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) activation. Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease. In this study, we investigated one mechanism of ENZ resistance, and tried to improve therapeutic efficiency of ENZ. We found HER2 expression is increased in ENZR tumors and cell lines, and is induced by ENZ treatment of LNCaP cells. ENZ-induced HER2 overexpression was dependent on AKT-YB1 activation and modulated AR activity. HER2 dependent AR activation in LNCaP and ENZR cells was effectively blocked by treatment with the EGFR/HER2 inhibitor Lapatinib, which reduced cell viability and increased apoptosis. Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth. However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC. These results suggest that while HER2 overexpression and subsequent AR activation is a targetable mechanism of resistance to ENZ, therapy using Lapatinib is only a rational therapeutic approach when used in combination with ENZ in CRPC.

Purpose: Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (ARFL) or variants (AR-Vs) in disease progression., Experimental Design: To define functional roles of ARFL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown ARFL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts., Results: ENZ-R-LNCaP cells express high levels of both ARFL and AR-V7 compared with CRPC-LNCaP; in particular, ARFL levels were approximately 12-fold higher than AR-V7. Both ARFL and AR-V7 are highly expressed in the nuclear fractions of ENZ-R-LNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of ARFL alone, or ARFL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and AR-regulated gene expression, and delayed tumor growth in vivo. In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both ARFL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of ARFL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts., Conclusions: These data identify the AR as an important driver of ENZ resistance, and while the contributions of ARFL and AR-Vs can vary across cell systems, ARFL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both ARFL and AR-Vs is a rational approach for AR-dependent CRPC., (©2015 American Association for Cancer Research.)

We define stress-induced adaptive survival pathways linking autophagy with the molecular chaperone clusterin (CLU) that function to promote anticancer treatment resistance. During treatment stress, CLU co-localizes with LC3 via an LIR-binding sequence within autophagosome membranes, functioning to facilitate LC3-Atg3 heterocomplex stability and LC3 lipidation, and thereby enhance autophagosome biogenesis and autophagy activation. Stress-induced autophagy is attenuated with CLU silencing in CLU(-/-) mice and human prostate cancer cells. CLU-enhanced cell survival occurs via autophagy-dependent pathways, and is reduced following autophagy inhibition. Combining CLU inhibition with anticancer treatments attenuates autophagy activation, increases apoptosis and reduces prostate cancer growth. This study defines a novel adaptor protein function for CLU under stress conditions, and highlights how co-targeting CLU and autophagy can amplify proteotoxic stress to delay cancer progression.

The progression to castration-resistant prostate cancer (CRPC) correlates with gain-of-function of the androgen receptor (AR) and activation of AKT. However, as single agents, AR or AKT inhibitors result in a reciprocal feedback loop. Therefore, we hypothesized that combination of an AKT inhibitor with an antiandrogen might result in a more profound, long-lasting remission of CRPC. Here, we report that the AKT inhibitor AZD5363 potently inhibits proliferation and induces apoptosis in prostate cancer cell lines expressing the AR and has anticancer activity in vivo in androgen-sensitive and castration-resistant phases of the LNCaP xenograft model. However, we found that the effect of castration-resistant tumor growth inhibition and prostate-specific antigen (PSA) stabilization is transient and resistance occurs with increasing PSA after approximately 30 days of treatment. Mechanistically, we found that single agent AZD5363 induces increase of AR binding to androgen response element, AR transcriptional activity, and AR-dependent genes such as PSA and NKX3.1 expression. These effects were overcome by the combination of AZD5363 with the antiandrogen bicalutamide, resulting in synergistic inhibition of cell proliferation and induction of apoptosis in vitro, and prolongation of tumor growth inhibition and PSA stabilization in CRPC in vivo. This study provides a preclinical proof-of-concept that combination of an AKT inhibitor with antiandrogen results in prolonged disease stabilization in a model of CRPC., (©2013 AACR.)

Although androgen receptor (AR) pathway inhibitors prolong survival in castrate-resistant prostate cancer (CRPC), resistance rapidly develops and is often associated with increased stress-activated molecular chaperones like clusterin (CLU) and continued AR signaling. Because adaptive pathways activated by treatment facilitate development of acquired resistance, cotargeting the stress response, activated by AR inhibition and mediated through CLU, may create conditional lethality and improve outcomes. Here, we report that CLU is induced by AR antagonism and silencing using MDV3100 and antisense, respectively, to become highly expressed in castrate- and MDV3100-resistant tumors and cell lines. CLU, as well as AKT and mitogen-activated protein kinase (MAPK) signalosomes, increase in response to MDV3100-induced stress. Mechanistically, this stress response is coordinated by a feed-forward loop involving p90rsk (RPS6KA)-mediated phosphoactivation of YB-1 with subsequent induction of CLU. CLU inhibition repressed MDV3100-induced activation of AKT and MAPK pathways. In addition, when combined with MDV3100, CLU knockdown accelerated AR degradation and repressed AR transcriptional activity through mechanisms involving decreased YB-1-regulated expression of the AR cochaperone, FKBP52. Cotargeting the AR (with MDV3100) and CLU (with OGX-011) synergistically enhanced apoptotic rates over that seen with MDV3100 or OGX-011 monotherapy and delayed CRPC LNCaP tumor and prostate-specific antigen (PSA) progression in vivo. These data indicate that cotargeting adaptive stress pathways activated by AR pathway inhibitors, and mediated through CLU, creates conditional lethality and provides mechanistic and preclinical proof-of-principle to guide biologically rational combinatorial clinical trial design.

Defining the mechanisms underlying metastatic progression of prostate cancer may lead to insights into how to decrease morbidity and mortality in this disease. An important determinant of metastasis is epithelial-to-mesenchymal transition (EMT), and the mechanisms that control the process of EMT in cancer cells are still emerging. Here, we report that the molecular chaperone Hsp27 (HSPB1) drives EMT in prostate cancer, whereas its attenuation reverses EMT and decreases cell migration, invasion, and matrix metalloproteinase activity. Mechanistically, silencing Hsp27 decreased IL-6-dependent STAT3 phosphorylation, nuclear translocation, and STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6-mediated EMT via modulation of STAT3/Twist signaling. We observed a correlation between Hsp27 and Twist in patients with prostate cancer, with Hsp27 and Twist expression each elevated in high-grade prostate cancer tumors. Hsp27 inhibition by OGX-427, an antisense therapy currently in phase II trials, reduced tumor metastasis in a murine model of prostate cancer. More importantly, OGX-427 treatment decreased the number of circulating tumor cells in patients with metastatic castration-resistant prostate cancer in a phase I clinical trial. Overall, this study defines Hsp27 as a critical regulator of IL-6-dependent and IL-6-independent EMT, validating this chaperone as a therapeutic target to treat metastatic prostate cancer., (©2013 AACR.)

TGF-β promotes epithelial-mesenchymal transition (EMT) and induces clusterin (CLU) expression, linking these genes to cancer metastasis. CLU is a pleiotropic molecular chaperone that confers survival and proliferative advantage to cancer cells. However, the molecular mechanisms by which TGF-β regulates CLU expression and CLU affects metastasis remain unknown. In this study, we report that the transcription factor Twist1 mediates TGF-β-induced CLU expression. By binding to E-boxes in the distal promoter region of CLU gene, Twist1 regulated basal and TGF-β-induced CLU transcription. In addition, CLU reduction reduced TGF-β induction of the mesenchymal markers, N-cadherin and fibronectin, thereby inhibiting the migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, our findings indicate that CLU is an important mediator of TGF-β-induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression.

Hsp27 is a stress-activated multifunctional chaperone that inhibits treatment-induced apoptosis and causes treatment resistance in prostate and other cancers. We previously showed that targeted suppression of Hsp27 sensitizes cancer cells to hormone and chemotherapy. However, mechanisms by which Hsp27 confers cell treatment resistance are incompletely defined. Here, we report that Hsp27 protects human prostate cancer cells against proteotoxic stress induced by proteasome inhibition, and that Hsp27 silencing using siRNA or antisense (OGX-427) induced both apoptosis and autophagy through mechanisms involving reduced proteasome activity and induction of endoplasmic reticulum (ER) stress. We found that autophagy activation protected against ER stress-induced cell death, whereas inhibition of autophagy activation following Hsp27 silencing using either pharmacologic inhibitors or atg3 silencing enhanced cell death. Importantly, cotargeting Hsp27 and autophagy by combining OGX-427 with the autophagy inhibitor, chloroquine, significantly delayed PC-3 prostate tumor growth in vivo. These findings identify autophagy as a cytoprotective, stress-induced adaptive pathway, activated following disruption of protein homeostasis and ER stress induced by Hsp27 silencing. Combinatorial cotargeting cytoprotective Hsp27 and autophagy illustrates potential benefits of blocking activation of adaptive pathways to improve treatment outcomes in cancer.

Clusterin is a stress-activated, cytoprotective chaperone that confers broad-spectrum treatment resistance in cancer. However, the molecular mechanisms mediating CLU transcription following anticancer treatment stress remain incompletely defined. We report that Y-box binding protein-1 (YB-1) directly binds to CLU promoter regions to transcriptionally regulate clusterin expression. In response to endoplasmic reticulum stress inducers, including paclitaxel, YB-1 is translocated to the nucleus to transactivate clusterin. Furthermore, higher levels of activated YB-1 and clusterin are seen in taxane-resistant, compared with parental, prostate cancer cells. Knockdown of either YB-1 or clusterin sensitized prostate cancer cells to paclitaxel, whereas their overexpression increased resistance to taxane. Clusterin overexpression rescued cells from increased paclitaxel-induced apoptosis following YB-1 knockdown; in contrast, however, YB-1 overexpression did not rescue cells from increased paclitaxel-induced apoptosis following clusterin knockdown. Collectively, these data indicate that YB-1 transactivation of clusterin in response to stress is a critical mediator of paclitaxel resistance in prostate cancer.

Small-molecule inhibitors of Hsp90 show promise in the treatment of castrate-resistant prostate cancer (CRPC); however, these inhibitors trigger a heat shock response that attenuates drug effectiveness. Attenuation is associated with increased expression of Hsp90, Hsp70, Hsp27, and clusterin (CLU) that mediate tumor cell survival and treatment resistance. We hypothesized that preventing CLU induction in this response would enhance Hsp90 inhibitor-induced CRPC cell death in vitro and in vivo. To test this hypothesis, we treated CRPC with the Hsp90 inhibitor PF-04929113 or 17-AAG in the absence or presence of OGX-011, an antisense drug that targets CLU. Treatment with either Hsp90 inhibitor alone increased nuclear translocation and transcriptional activity of the heat shock factor HSF-1, which stimulated dose- and time-dependent increases in HSP expression, especially CLU expression. Treatment-induced increases in CLU were blocked by OGX-011, which synergistically enhanced the activity of Hsp90 inhibition on CRPC cell growth and apoptosis. Accompanying these effects was a decrease in HSF-1 transcriptional activity as well as expression of HSPs, Akt, prostate-specific antigen, and androgen receptor. In vivo evaluation of the Hsp90 inhibitors with OGX-011 in xenograft models of human CRPC showed that OGX-011 markedly potentiated antitumor efficacy, leading to an 80% inhibition of tumor growth with prolonged survival compared with Hsp90 inhibitor monotherapy. Together, our findings indicate that Hsp90 inhibitor-induced activation of the heat shock response and CLU is attenuated by OGX-011, with synergistic effects on delaying CRPC progression., (©2011 AACR.)

Signal transducer and activator of transcription 5 (Stat5) plays an important role in the transition of prostate cancer (PCa) to its castrate-resistant state. Pharmacologic targeting of Stat5 is a rational approach to delay castrate-resistant progression, in part, because Stat5 cooperates with the androgen receptor (AR) to promote PCa progression. Immunostaining of tissue microarrays was used to correlate Stat5 expression with Gleason grade and to characterize changes in treatment-naive and androgen-deprived human PCa. Potency of a Stat5 antisense oligonucleotide (ASO) on Stat5 knockdown, cell growth, and apoptosis was assessed in LNCaP, C4-2, and DU145 cells. Effects of Stat5 knockdown on AR activity and stability was assessed using a PSA transactivation-luciferase assay and cyclohexamide plus MG132 treatment, respectively. LNCaP tumor-bearing mice were castrated and randomly assigned to treatment with Stat5-ASO or controls. Here, we show that the frequency of Stat5 expression is significantly increased in high Gleason grade as well as in hormone-treated PCa. Also, specific knockdown of Stat5 with ASO abrogates androgen-induced AR nuclear translocation and PSA transactivation despite R1881 stimulation. Moreover, Stat5 knockdown destabilizes AR, which leads to AR degradation via the proteasome. Shown for the first time as a preclinical proof-of-principle, Stat5 knockdown with Stat5-ASO significantly delays CRPC tumor progression in vivo. Thereby, we are able to recapitulate our in vitro results by reducing serum PSA and expression levels of target proteins in the xenograft tumors.

Hsp27 is highly expressed in castrate-resistant prostate cancer. Although its overexpression confers resistance to androgen ablation and chemotherapy, the mechanisms by which Hsp27 inhibits treatment-induced apoptosis are incompletely defined. Castrate-resistance often correlates with increased activity of autocrine and/or paracrine growth/survival stimulatory loops including the mitogen-activated protein kinase (MAPK) and Akt pathways and insulin-like growth factor (IGF) axis components. Because Hsp27 can be activated by both MAPK and Akt pathways, it is possible that interactions between IGF-I signaling and Hsp27 phosphoactivation function to promote castrate-resistant progression. Here, we report that Hsp27 expression and phosphorylation levels correlate with IGF-I signaling and castrate-resistant progression in human prostate cancer specimens and cell lines. IGF-I induces Hsp27 phosphorylation in a time- and dose-dependent manner via p90Rsk, which interacts directly with and phosphorylates Hsp27 in vitro and in vivo. Conversely, p90Rsk inhibition using short interfering RNA or a dominant negative mutant abolishes IGF-I-induced Hsp27 phosphorylation. Hsp27 overexpression increases IGF-I-induced phosphorylation of Erk, p90Rsk, and Akt. Conversely, Hsp27 knockdown abrogates IGF-I-induced phosphorylation of Erk, p90Rsk, and Akt, thereby destabilizing Bad/14-3-3 complexes and increasing apoptotic rates. These data elucidate the interactions between Hsp27 phosphorylation and the IGF-I receptor signaling pathway and support targeting Hsp27 as a therapeutic strategy for castrate-resistant prostate cancer.

Secretory clusterin (sCLU) is a stress-activated, cytoprotective chaperone that confers broad-spectrum cancer treatment resistance, and its targeted inhibitor (OGX-011) is currently in phase II trials for prostate, lung, and breast cancer. However, the molecular mechanisms by which sCLU inhibits treatment-induced apoptosis in prostate cancer remain incompletely defined. We report that sCLU increases NF-kappaB nuclear translocation and transcriptional activity by serving as a ubiquitin-binding protein that enhances COMMD1 and I-kappaB proteasomal degradation by interacting with members of the SCF-betaTrCP E3 ligase family. Knockdown of sCLU in prostate cancer cells stabilizes COMMD1 and I-kappaB, thereby sequestrating NF-kappaB in the cytoplasm and decreasing NF-kappaB transcriptional activity. Comparative microarray profiling of sCLU-overexpressing and sCLU-knockdown prostate cancer cells confirmed that the expression of many NF-kappaB-regulated genes positively correlates with sCLU levels. We propose that elevated levels of sCLU promote prostate cancer cell survival by facilitating degradation of COMMD1 and I-kappaB, thereby activating the canonical NF-kappaB pathway.

Androgen receptor (AR) transactivation is known to enhance prostate cancer cell survival. However, the precise effectors by which the prosurvival effects of androgen and AR drive prostate cancer progression are poorly defined. Here, we identify a novel feed-forward loop involving cooperative interactions between ligand-activated AR and heat-shock protein 27 (Hsp27) phospho-activation that enhance AR stability, shuttling, and transcriptional activity, thereby increasing prostate cancer cell survival. Androgen-bound AR induces rapid Hsp27 phosphorylation on Ser(78) and Ser(82) residues in an AR- and p38 kinase-dependent manner. After this androgen-induced, non-nuclear phospho-activation, Hsp27 displaces Hsp90 from a complex with AR to chaperone AR into the nucleus and interact with its response elements to enhance its genomic activity. Inhibition of Hsp27 phosphorylation, or knockdown using the antisense drug OGX-427, shifted the association of AR with Hsp90 to MDM2, increased proteasome-mediated AR degradation, decreased AR transcriptional activity, and increased prostate cancer LNCaP cell apoptotic rates. OGX-427 treatment of mice bearing LNCaP xenografts transfected with an androgen-regulated, probasin-luciferase reporter construct resulted in decreased bioluminescence and serum PSA levels as pharmacodynamic readouts of AR activity, as well as AR, Hsp27, and Hsp90 protein levels in LNCaP tumor tissue. These data identify novel nongenomic mechanisms involving androgen, AR, and Hsp27 activation that cooperatively interact to regulate the genomic activity of AR and justify further investigation of Hsp27 knockdown as an AR disrupting therapeutic strategy in prostate cancer.

Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by >90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer.

One strategy to improve therapies in prostate cancer involves targeting cytoprotective genes activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. The objectives of this study were to define changes in Hsp27 levels after androgen ablation and to evaluate the functional relevance of these changes in AI progression. Using a tissue microarray of 232 specimens of hormone-naïve and post-hormone ablation-treated prostate cancer, we found that Hsp27 levels increase after androgen ablation to become highly expressed (>4-fold, P < or = 0.01) in AI tumors. Hsp27 overexpression rendered LNCaP cells highly resistant to androgen withdrawal both in vitro and in vivo. Tumor volume and serum prostate-specific antigen levels increased 4.3- and 10-fold faster after castration when Hsp27 was overexpressed. Treatment of LNCaP tumor cells in vitro with Hsp27 antisense oligonucleotides (ASO) or short-interfering RNA suppressed Hsp27 levels in a dose-dependent and sequence-specific manner increased the apoptotic sub-G0-G1 fraction and caspase-3 cleavage >2-fold, as well as decreased signal transducers and activators of transcription 3 (Stat3) levels and its downstream genes, c-fos and sPLA-2. The cytoprotection afforded by Hsp27 overexpression was attenuated by Stat3 knockdown using specific Stat3 ASO. Coimmunoprecipitation and immunofluorescence confirmed that Hsp27 interacts with Stat3 and that Stat3 levels correlated directly with Hsp27 levels. Hsp27 ASO treatment in athymic mice bearing LNCaP tumors significantly delayed LNCaP tumor growth after castration, decreasing mean tumor volume and serum prostate-specific antigen levels by 57% and 69%, respectively. These findings identify Hsp27 as a modulator of Stat3-regulated apoptosis after androgen ablation and as a potential therapeutic target in advanced prostate cancer.

Heat shock protein 27 (Hsp27) is a chaperone implicated as an independent predictor of clinical outcome in prostate cancer. Our aim was to characterize changes in Hsp27 after androgen withdrawal and during androgen-independent progression in prostate xenografts and human prostate cancer to assess the functional significance of these changes using antisense inhibition of Hsp27. A tissue microarray was used to measure changes in Hsp27 protein expression in 232 specimens from hormone naive and posthormone-treated cancers. Hsp27 expression was low or absent in untreated human prostate cancers but increased beginning 4 weeks after androgen-ablation to become uniformly highly expressed in androgen-independent tumors. Androgen-independent human prostate cancer PC-3 cells express higher levels of Hsp27 mRNA in vitro and in vivo, compared with androgen-sensitive LNCaP cells. Phosphorothioate Hsp27 antisense oligonucleotides (ASOs) and small interference RNA potently inhibit Hsp27 expression, with increased caspase-3 cleavage and PC3 cell apoptosis and 87% decreased PC3 cell growth. Hsp27 ASO and small interference RNA also enhanced paclitaxel chemosensitivity in vitro, whereas in vivo, systemic administration of Hsp27 ASO in athymic mice decreased PC-3 tumor progression and also significantly enhanced paclitaxel chemosensitivity. These findings suggest that increased levels of Hsp27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression.

Introduction: Lung cancer is highly lethal and resistant to most anticancer interventions. Treatment resistance is mediated, in part, by enhanced expression of cell survival proteins that help facilitate tumor progression. Clusterin is a stress-associated cytoprotective protein up-regulated by various apoptotic triggers in many cancers and confers treatment resistance when overexpressed. The objectives in this study were to evaluate clusterin expression levels in human lung cancer tissue, and to test effects of clusterin silencing using antisense oligonucleotides (ASOs) and short interfering double-stranded RNAs (siRNAs) on chemosensitivity in human lung cancer A549 cells., Methods: Clusterin immunostaining was evaluated in a tissue microarray of 149 spotted human lung cancers. The effects of clusterin ASO or siRNA treatment on clusterin expression and chemosensitivity to paclitaxel was examined in A549 cells in vitro while the ability of clusterin ASO to chemosensitize in vivo was evaluated in immunocompromised mice bearing A549 tumors., Results: More than 80% of human non-small cell lung cancers are immunoreactive for clusterin. Clusterin ASO or siRNA decreased clusterin mRNA expression in A549 cells >75% in a dose-dependent, sequence-specific manner, and significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo administration of clusterin ASO, compared to mismatch control oligonucleotide, synergistically enhanced the effects of paclitaxel or gemcitibine to significantly delay A549 tumor growth., Conclusion: These findings identify clusterin as a valid therapeutic target in strategies employing novel multimodality therapy for advanced lung cancer.

Research investigating the molecular events underlying progression of prostate cancer to androgen independence has been impeded by the lack of an appropriate in vivo model that yields "pure" populations of prostate cancer cells that are not contaminated with host cells. Here we characterize a new in vivo model that uses hollow fibers and allows for the retrieval of uncontaminated prostate cancer cells during various stages of endocrine progression to androgen independence in male immunocompromised mice. Prostate-specific antigen (PSA) gene expression, proliferation of cells, and histology were examined in these mice before and after castration. LNCaP cells seeded at a density of 1 x 10(7) cells/ml, or a total of approximately 4.8 x 10(6) cells/animal, provided measurable serum PSA levels that increased in intact (noncastrated) animals, decreased by 80% to a nadir after castration, and subsequently increased by 4 weeks after castration, indicating progression to androgen independence. In vivo proliferation of LNCaP cells inside the fibers continued in the presence of androgens and continued to increase, albeit at a slower rate, in the castrated animals. Histology of cells cultivated in hollow fibers demonstrated that initially the cells grew along the wall of the fiber and tended to stack up, forming layers and scaffold structures resembling a solid tumor. Fibers removed from castrated animals with elevated levels of serum PSA contained spheroids of cells that had detached from the fiber wall. The development of the LNCaP hollow fiber model described here provides a reproducible means of obtaining "pure" populations of LNCaP cells during different stages of progression to androgen independence for molecular analysis requiring RNA and protein extracts free of host cell contamination.

Most patients that present in the clinic with prostate cancer have either localized or recurrent postradiotherapy therapy tumors that may be amenable to injectable treatments using slow-release cytotoxic drugs. The objective of this preclinical study was to design an injectable polymeric paste formulation of paclitaxel for intratumoral injection into nonmetastatic human prostate tumors grown s.c. in mice. Paclitaxel was dissolved (10% w/w) in a blend of a biodegradable triblock copolymer of a random copolymer of D,L-lactide and epsilon-caprolactone (PLC) with poly(ethyleneglycol) [PEG; PLC-PEG-PLC] blended with methoxypoly(ethylene glycol) in a 40:60 ratio. Human prostate LNCaP tumors grown s.c. in castrated athymic male mice were injected with 100 microl of this paste at room temperature. Changes in tumor progression were assessed using both serum prostate-specific antigen (PSA) levels and tumor size. Paclitaxel inhibited LNCaP cell growth in vitro in a concentration-dependent fashion with an IC50 of 1 nM. Apoptosis was documented using DNA fragmentation analysis. The paste formulation solidified over a period of 1 h both in vivo and in aqueous media at 37 degrees C as the methoxypoly(ethylene glycol) component partitioned out of the insoluble PLC-PEG-PLC/paclitaxel matrix. The semisolid implant released drug at a rate of about 100 microg/day in vitro. In control mice treated with paste without paclitaxel, serum PSA levels increased from 2-8 ng/ml (mean, 4.3+/-2 ng/ml) to 60-292 ng/ml (mean, 181+/-88 ng/ml), and tumor volume increased from 30 to 1000 mm3. In mice treated with a single 100-microl injection 3 weeks after castration (early-phase treatment group), tumors decreased in volume from a mean of 43+/-19 mm3 to nonpalpable, and PSA levels decreased from a mean of 22+/-8 to 2+/-1 ng/ml by 8 weeks after castration. In mice treated 5 weeks after castration (androgen-independent tumors; late-phase treatment group), tumors decreased in volume from a mean of 233+/-136 mm3 to nonpalpable, and serum PSA decreased from 24+/-8 to 9+/-4 ng/ml. Observed side effects of the treatment were limited to minor ulceration at the needle injection site in paclitaxel-treated mice only. The controlled-release formulation can be injected via 22-gauge needles and is effective in inhibiting LNCaP tumor growth and PSA levels in mice bearing multiple nonmetastatic tumors. Paclitaxel may be an effective therapy for patients with localized tumors recurring after radiotherapy and for some patients with localized tumors who are not candidates for radical treatment.

Bcl-2 has emerged as a critical regulator of apoptosis in a variety of cell systems and is up-regulated during progression to androgen independence in prostate cancer cells. The objectives of this study were to characterize changes in Bcl-2 after androgen withdrawal and during progression to androgen independence in the human prostate LNCaP tumor model and determine whether adjuvant use of antisense Bcl-2 oligodeoxynucleotides (ODNs) with androgen ablation delays progression to androgen independence. Bcl-2 expression in LNCaP cells is down-regulated to undetectable levels by androgen in vitro and up-regulated after castration in vivo. Antisense Bcl-2 ODN treatment reduced LNCaP cell Bcl-2 messenger RNA and protein levels by >90% in a sequence-specific and dose-dependent manner at concentrations >50 nM. Bcl-2 mRNA levels returned to pretreatment levels by 48 h after discontinuing treatment. Athymic male mice bearing SQ LNCaP tumors were castrated and injected i.p. with 12.5 mg/kg/day with two-base mismatch ODN control, reverse polarity ODN control, or antisense Bcl-2 ODN. Tumor volume in control mice gradually increased 5-fold (range, 3-6) by 12 weeks after castration compared to a 10-50% decrease in precastrate tumor volume in mice treated with antisense Bcl-2 ODN. Changes in serum PSA paralleled changes in tumor volume, increasing 4-fold faster above nadir in controls than in mice treated with antisense Bcl-2 ODN. After decreasing 70% by 1 week after castration, PSA increased 1.6-fold above precastrate levels by 11 weeks in controls while staying 30% below precastrate levels in antisense-treated mice. In a second group of experiments, LNCaP tumor growth and serum PSA levels were 90% lower (P<0.01) in mice treated with antisense Bcl-2 ODN compared with mismatch or reverse polarity ODN controls. These results support the hypothesis that Bcl-2 helps mediate progression to androgen independence and is an appropriate target for antisense therapy.

Several metastasizing murine and human animal models for prostate cancer are available. However, these models are androgen-independent and lack differentiated features such as androgen receptor and androgen-regulated gene expression like prostate-specific antigen (PSA). The objective of this study was to develop a metastasizing prostate cancer model with differentiated features using the human LNCaP cell line. Athymic and SCID mice were injected either s.c. or intraprostatically with 1 x 10(6) LNCaP cells. Changes in serum and tumor PSA mRNA levels were determined before and after castration to assess time to androgen-independent progression. Local tumor and metastatic growth was assessed at sacrifice after 12 weeks. Reverse transcription-PCR (RT-PCR) was used to detect circulating LNCaP cells. LNCaP tumor incidence after s.c. injection was 100% (65 of 65) in SCID mice and 80% in athymic mice. No lymph node or distant metastases were observed with s.c. tumors, and RT-PCR for PSA transcripts was negative. Primary tumor incidence after intraprostatic injection was 89% (39 of 44) in SCID mice and 60% in athymic mice. In 10 SCID mice with primary tumors followed for 12 weeks, retroperitoneal or mediastinal lymph node metastases were found in 100%, and microscopic pulmonary metastases were identified in 40%. RT-PCR for PSA transcripts was positive in 3 of 10 mice tested. Serum PSA levels in mice with s.c. and intraprostatic tumors decreased by 65% to nadir levels at 7 and 4 days after castration, respectively. Serum PSA and LNCaP tumor PSA mRNA levels increased to precastration levels earlier in SCID mice with intraprostatic tumors compared to those with s.c. tumors. Intraprostatic injection of LNCaP cells in SCID mice provides a useful animal model to investigate mechanisms of metastasis and to evaluate therapies targeted toward inhibiting the metastatic cascade.

Progesterone, 17-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone and oestradiol concentrations in the plasma were measured by simultaneous radioimmunoassay in males of the lizard Podarcis s. sicula. Hormonal determinations were performed at monthly intervals from January to December (except for August). Testosterone and androstenedione reached peak values of 174.8 ng/ml and 21.4 ng/ml in the mating season (spring) and then testosterone fell abruptly to 5.9 ng/ml in June remaining at this level during hibernation when dehydroepiandrosterone (DHA) reached a maximal level of 28.5 +/- 9.3 ng/ml. Castration resulted in a marked decrease of testosterone, androstenedione, dihydrotestosterone and DHA values, with DHA being significantly lowered only during the winter season. In castrated animals, however, testosterone and androstenedione persisted conspicuously in the plasma during the breeding period, suggesting that adrenal sex steroid output may change during the annual reproductive cycle. In intact animals, progesterone and oestradiol exhibited peak values during the refractory period after the mating season. We suggest a probable role of oestradiol in the induction of the refractory period in this lizard.

Leydig cell function was studied in 108 varicocele (V) patients with a mean age of 30.9 years, and a control group (C) of 46 men with a mean age of 30 years. Plasma gonadotropin levels were determined before and after GNRH stimulation. Testosterone (T), 17-OH-progesterone (17-OH-P), dihydrotestosterone (DHT) and estradiol (E2) were also assayed. Mean plasma T levels were significantly decreased in varicocele patients (V = 416 +/- 12.9, n = 106; C = 487 +/- 19.9, n = 40; P less than 0.01), while the basal 17-OH-P/T ratio was significantly increased (V = 0.38 +/- 0.02, n = 56; C = 0.28 +/- 0.02, n = 40; 0.02 greater than P greater than 0.01) and remained higher after hCG stimulation (P less than 0.01). No significant differences in mean sex steroid levels were observed when comparing varicocele patients with normal sperm counts (VN) and those who had oligozoospermia (VO). There was a significant negative linear correlation between age and 17-OH-P (n = 56; r = -0.47; P less than 0.01) and T values (n = 106; r = 0.27; P less than 0.01) in varicocele patients, which contrasted with the absence of any significant correlation with age in the controls. These data suggest that the duration of idiopathic varicocele influences testicular hormone secretion.